Available online at www.sciencedirect.com

Nuclear Medicine and Biology 36 (2009) 985 – 991
www.elsevier.com/locate/nucmedbio

Intracellular reactions affecting 2-amino-4-([11 C]methylthio)butyric acid([11 C]methionine) response to carbon ion radiotherapy in C10 glioma cells
Tomoya Ueharaa,⁎, Misuzu Nakagawaa , Nobuhiko Takaib , Sachiko Koikec , Yoshiya Furusawac ,Kouichi Andoc , Keiichi Kawaid , Hiromichi Akizawae , Toshiaki Iriec , Yasushi AranoaaGraduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan
Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo 859-3298, Japanc
National Institute of Radiological Sciences, Chiba 263-8555, JapandSchool of Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa 920-0942, Japane
Health Sciences University of Hokkaido, Ishikari-gun, Hokkaido 061-0293, Japan
Received 28 February 2009; received in revised form 22 June 2009; accepted 23 July 2009b

Abstract
Purpose: The response of 2-amino-4-([14C]methylthio)butyric acid ([14C]Met) uptake and [125I]3-iodo-alpha-methyl-L-tyrosine ([125I]IMT)
uptake to radiotherapy of C10 glioma cells was compared to elucidate the intracellular reactions that affect the response of 2-amino-4-([11C]
methylthio)butyric acid ([11C]Met) uptake to radiotherapy.
Methods: After irradiation of cultured (3 Gy) or xenografted C10 glioma cells (25 Gy) using a carbon ion beam, the accumulation of [14C]
Met and [125I]IMT in the tumors was investigated. The radiometabolites in xenografted tumors after radiotherapy were analyzed by sizeexclusion HPLC.
Results: [14C]Met provided earlier responses to the carbon ion beam irradiation than [125I]IMT in both cultured and xenografted tumors.
While [125I]IMT remained intact in xenografted tumor before and after irradiation, the radioactivity derived from [14C]Met was observed
both in high molecular fractions and intact fractions, and the former decreased after irradiation.
Conclusion: The earlier response of [11C]Met uptake to tumor radiotherapy could be attributable to the decline in the intracellular energydependent reactions of tumors due to radiotherapy.
© 2009 Elsevier Inc. All rights reserved.
Keywords: Radiotherapy; Carbon-ion; 11C-methionine; Cellular activity

1. Introduction
Carbon ion radiotherapy started at the National Instituteof Radiological Sciences (NIRS, Japan) using Heavy Ion
Medical Accelerator in Chiba (HIMAC) synchrotron
accelerator in 1994. So far, HIMAC at NIRS has treatedover 3000 patients suffering from malignant disease [1–3].
Although a carbon ion beam has higher relative biologicaleffectiveness and smaller differences in sensitivity between
cell lines, the responses of tumor cells after irradiation using
a carbon ion beam still differed between cells [4]. This calls
for a precise and reliable method for estimating the⁎ Corresponding author. Tel.: +81 43 226 2898; fax: +81 43 226 2897.
E-mail address: tuehara@p.chiba-u.ac.jp (T. Uehara).
0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.07.005

therapeutic efficacy of carbon ion beams at an early stageof the treatment.
In general, morphological diagnosis, such as X-ray
computed tomography (CT) and magnetic resonance imaging (MRI), are used to assess the response of cancers totherapy [5]. However, morphological diagnosis is affectedby the differentiation of viable tumor cells from necrosis/scartissue. Moreover, it is necessary to wait for a long period
after radiotherapy before an accurate evaluation can be made[6,7]. On the other hand, functional imaging with positronemission tomography (PET) using radiopharmaceuticals
such as 2-[18F]-fluoro-2-deoxy-D-glucose ([18F]FDG) or 2amino-4-([11C]methylthio)butyric acid ([11C]Met, Fig. 1A)
can elucidate biochemical and physiological changes intumor cells before morphological changes occur in the tumor

986

T. Uehara et al. / Nuclear Medicine and Biology 36 (2009) 985–991

pared after irradiation of cultured C10 glioma cells or thesame cells xenografted into nude mice using a carbon ion
beam. The critical cellular reactions responsible for the
response of tumors to [11 C]Met accumulation afterradiotherapy are discussed.
2. Materials and methods
2.1. GeneralFig. 1. Structures of Met (A) and IMT (B).

[8,9]. This imaging technique has been applied to estimate
tumor responses to radiotherapy [7,10–13].
It is well known that irradiation induces an attraction andactivation of inflammatory cells such as macrophages[13,14]. These inflammatory cells lead to a prominent
increase in some radiopharmaceuticals such as [18F]FDG
comparable with that in tumor cells [13,14]. The radiopharmaceuticals that accumulated in the inflammationinduced by irradiation made it difficult to predict theefficacy of tumor radiotherapy [13,14]. The lower accumulation of [11C]Met at sites of irradiation compared with [18F]
FDG renders [11C]Met as the radiopharmaceutical of choicefor this purpose [13,14]. However, because cyclotron
facilities are required to produce and use [11C]Met, due toits short half-life (20 min), the development of 18F (T1/2: 110min) or 123I (T1/2: 13 h)-labeled radiopharmaceuticals thatafford diagnostic information similar to that of [11C]Met ishighly desirable.
The accumulation of [11C]Met in tumors is mediated byseveral cellular reactions, such as amino acid transport acrossthe membrane, intracellular protein synthesis and transmethylation [9]. However, it remains uncertain which
cellular reactions are directly correlated with the reductionin [11C]Met accumulation after radiotherapy. The answer tothis question paves the way for designing 18F- or 123 I-labeled
radiopharmaceuticals that afford diagnostic informationcomparable to [11C]Met. The uptake of [11C]Met by tumors
is mediated predominantly by energy independent aminoacid transport system L [15,16], whereas protein synthesis
and trans-methylation are energy dependent reactions [17].
The artificial amino acid [123 I]3-iodo-alpha-methyl-L-tyrosine ([123I]IMT, Fig. 1B) is also transported to tumor cells byamino acid transport system L [18–21], whereas [123I]IMT is
not involved in protein synthesis [22–24], indicating that theuptake of [123 I]IMT into tumors only reflects the activity ofamino acid transport system L. This suggested that thecomparative tumor accumulation of [11C]Met and [123I]IMT
could provide insights for understanding the cellular
reactions correlated with the response of tumors to [11C]
Met accumulation after radiotherapy.
In the present study, tumor accumulation and intracellular metabolism of [14C]Met and [125 I]IMT were com-

2-Amino-4-([14C]methylthio)butyric acid ([14C]Met) was
purchased from GE Healthcare Bioscience (Tokyo, Japan).
Size-exclusion high-performance liquid chromatography(SE-HPLC) was performed using a Cosmosil 5Diol-300
column (7.5×600 mm, Nacalai Tesque, Kyoto, Japan) eluted
with 0.1 M phosphate buffer (pH 6.8) at a flow rate of 1.0 ml/
min. Each eluent was collected with a fraction collector(RadiFlac, GE Healthcare Bioscience) at 1-min intervals,
and the radioactivity counts in each fraction (1 ml) were
determined with an auto well gamma counter (ARC-380M,
Aloka, Tokyo, Japan). 14C-Radioactivity was measured witha liquid scintillation counter (LSC-5100, Aloka). [125I]IMT
was prepared according to the procedure described previously [24]. The C10 glioma cell line was provided from the
National Institute of Radiological Sciences (NIRS, Chiba,
Japan). Other reagents were of reagent grade and usedwithout further purification.
2.2. Cell culture
C10 Glioma cells were grown as a monolayer in
McCoy's 5A medium (Invitrogen, Tokyo, Japan) supplemented with 10% fetal bovine serum (SAFC Biosciences,
Kansas City, MO, USA), penicillin (100 U/ml), andstreptomycin (100 μg/ml) at 37°C in an atmosphere of 5%
CO2. Before the cell uptake experiments, C10 glioma cells
were washed twice with phosphate-buffered saline (PBS)
and trypsinized using a solution of 0.05% trypsin in PBS.
After the cells were resuspended in McCoy's 5A medium,the cell number was counted by a Coulter counter (Beckman
Coulter KK, Tokyo, Japan).
2.3. Irradiation
Carbon ion beams were accelerated up to 290 MeV/U
with the Heavy Ion Medical Accelerator in Chiba (HIMAC)
synchrotron at NIRS. Modulator films were used to performa LET value of 60 keV/μm with a spread-out Bragg peak of
6-cm width. The sample was irradiated at room temperature.
2.4. In vitro experiments
Cell uptake experiments were performed according to theprocedure of Samnick et al. [25] with slight modifications.
The cell suspension was transferred to 2-ml Eppendorf tubes
at a concentration of 7×105 cells. After the cells were
centrifuged for 5 min at 300×g, the resulting supernatant wasremoved, and the pellet was washed twice with 2 ml of

T. Uehara et al. / Nuclear Medicine and Biology 36 (2009) 985–991

HEPES buffer (pH 7.4, 12 mM HEPES, 137 mM NaCl, 2.7
mM KCl, 1.0 mM MgCl2, and 5.6 mM D-glucose). For the
Na+-free uptake study, choline-Cl replaced NaCl. The cells
were resuspended in HEPES buffer (500 μl) and preincubated for 15 min at 37°C to equilibrate the cells. Aliquots of
20 μl ([125I]IMT: 11.1 kBq, 0.14 pmol, [14C]Met: 3.7 kBq,
1.6 nmol) were added to the cells in the presence or absenceof an inhibitor for amino acid transporter system A or L [5mM alpha-(methylamino) isobutyric acid (MeAIB) or 5 mM
2-amino-2-norbornane carboxylic acid (BCH), respectively],
and the cells were incubated at 37°C for 3 min. Afterterminating the tracer uptake reaction using 1 ml of ice-cold
PBS and a subsequent 2-min incubation in an ice bath, thecells were centrifuged for 2 min at 300×g. The resulting
supernatant was removed, and the pellet was washed threetimes with ice-cold PBS (1 ml). 125I-Radioactivity counts inthe cells were measured with a gamma counter, while 14Cradioactivity levels were determined with a liquid scintillation counter (LSC-6000, Aloka) after cells were dissolved intissue solubilizer (0.5 ml, SOLUENE-350, Packard Japan,
Tokyo, Japan) and scintillator (5 ml, HYONIC-FLUOR,
Packard Japan). The data were expressed as uptake per
million cells. Each assay was performed in triplicate.
2.5. Cell uptake experiments after irradiation
The cells (5.6×105 cells) were incubated in T-25 flasks(Nalge Nunc, Tokyo, Japan) for 4 days. Three to four flasks
were used for each assay. The flasks were irradiated by acarbon ion beam (3 Gy) at room temperature. The cell
survival rate after carbon ion beam irradiation (3 Gy) was
about 30% of nonirradiated cells (data not shown). After
irradiation, the medium was exchanged for a fresh medium.
The cellular uptake of [125I]IMT (11.1 kBq, 5 nmol) or [14C]
Met (3.7 kBq, 5 nmol) by C10 glioma cells was investigatedfrom 1 to 4 days after irradiation in accordance with theabove procedure. The data were expressed as uptake per
million cells. Each assay was performed in triplicate.

987

5 nmol) dissolved in 100 μl of saline were simultaneously
administered intravenously before sacrifice by decapitation
after 1 h. The difference in the specific activities of theseradiopharmaceuticals could be ignored because the methionine concentration in the serum of the mice is high (ca. 300μM) [26]. Tissues of interest were removed and weighed,and the 125I-radioactivity counts were determined using an
auto well gamma counter (Aloka). The tissue was thendissolved in soluene-350 (0.5 ml, Packard Japan) andHionic-Fluor (4 ml, Packard Japan). The 14C-radioactivity
counts were measured with a liquid scintillation counter(Aloka). 14 C-Radioactivity counts were determined bysubtracting the 125 I-radioactivity counts from the total
count. On the other hand, to evaluate the accumulation of[125I]IMT and [14C]Met at the irradiated site, the left hindlegs (muscle) without tumors were also irradiated using thecarbon ion beam (25 Gy) and investigated by the sameprocedure as above.
2.7. Metabolic studies in tumor
C10 Glioma cells (1×106 cells) were transplanted
subcutaneously into the left hind legs of nude mice 7 days
before the irradiation. The tumor-containing left hind legs
were irradiated by the carbon ion beam (25 Gy) as describedabove. Two days after irradiation, [125 I]IMT (740 kBq, 5pmol) and [14C]Met (740 kBq, 0.17 μmol) dissolved in 100

2.6. In vivo experiments
Animal studies were conducted in accordance with theinstitutional guidelines and were approved by the Chiba
University Animal Care Committee. Four to five mice foreach point were used. C10 Glioma cells (1×106 cells) weretransplanted subcutaneously into the left hind legs of nude
mice 7 days before irradiation. Under pentobarbital anesthesia (20 mg/kg), the mice were immobilized on a Lucite plate
to place their left hind legs in the irradiation field. The tumorcontaining left hind legs received carbon ion beamirradiation (25 Gy). No tumor regrowth was observed forone month after carbon ion radiotherapy (25 Gy) (data notshown). Tumor diameters were measured by externalmeasurement in three dimensions using a caliper, andtumor volumes (v) were determined using the formulav=a×b×c (where a, b and c represent the tumor diameter).
[125 I]IMT (11.1 kBq, 0.14 pmol) and [14C]Met (11.1 kBq,

Fig. 2. The uptake of [125I]IMT (A) and [14C]Met (B) by C10 glioma cells.
Values are mean±S.D. MeAIB, an inhibitor of amino acid transporter systemA, showed no significant inhibitory effect on [125I]IMT or [14C]Met uptake.
An inhibitor of amino acid transporter system L, BCH, significantly reduced
the uptake of both [125I]IMT (98%) and [14C]Met (82%).

988

T. Uehara et al. / Nuclear Medicine and Biology 36 (2009) 985–991

μl of saline were simultaneously administered intravenously
to mice before being sacrificed by decapitation after 1 h.
Tumor tissues were removed and weighed. Tumor samples(0.5 g) were placed in a test tube and subjected to threecycles of freezing (dry ice-methanol bath) and thawing. Afterthe addition of 5 volumes of 0.1 M Tris-buffer (pH 6.5)
containing 0.15 M NaCl, 0.02% sodium azide, 1 trypsin
inhibitor unit/ml aprotinin, 2 mM benzamide-HCl, 2 mM
iodoacetamide, 1 mM phenylmethanesulfonyl fluoride,
5 mM diisopropyl fluorophosphates and 35 mM β-octylglucoside, the samples were homogenized using a Polytron

homogenizer (PT10-35, Kinematica, Littau, Switzerland) at
full speed with three consecutive 30-s bursts with incubation
on ice before centrifugation at 48000×g for 20 min at 4°C(Optima MAX, Beckman Coulter KK). The supernatant was
separated from the pellet. The tumor supernatant was
analyzed by SE-HPLC after filtration through a polycarbonate membrane with a pore diameter of 0.45 μm (Nacalai
Tesque). Two mice for each point were used, and the data
were expressed as the average of the two experiments.
2.8. Statistical evaluation
Unpaired Student's t test was used for the experiments.
Results were considered statistically significant at Pb.05.

Fig. 3. Time course of changes in the cell numbers [control (circle),
irradiation (square)] per flask (A), [125I]IMT uptake (B) and [14C]Met uptake(C) by C10 glioma cells after irradiation using the carbon ion beam (3 Gy).
Values are mean±S.D. (n=3). Significant differences were determined byunpaired Student's t test. #Pb.05 vs. control; *Pb.05 vs. Day 0.

Fig. 4. Time course of changes in tumor volume (A), [125I]IMT the uptake(B) and [14C]Met uptake (C) in xenografted C10 glioma-bearing nude mice
after irradiation using the carbon ion beam (25 Gy). Values are mean±S.D.
(n=4–5). Significant differences were determined by unpaired Student's t
test. *Pb.05 vs. Day 0.

T. Uehara et al. / Nuclear Medicine and Biology 36 (2009) 985–991

989

3. Results
3.1. In vitro experiments
The tumor uptake of [125 I]IMT was not reduced in thepresence of MeAIB, an inhibitor of amino acid transportsystem A, but was almost completely (98%) inhibited in thepresence of BCH, an inhibitor of amino acid transporter
system L (Fig. 2). In Na+-free buffer, [125 I]IMT uptake was
reduced to 21% of the control. [14C]Met uptake was notinhibited by MeAIB but was reduced by BCH to 82% of thecontrol. In Na+-free buffer, [14C]Met uptake was reduced to12% (Fig. 2).
The time course of changes in cell number after
irradiation using the carbon ion beam (3 Gy) is shown in
Fig. 3. The cell number increased until 3 days after
irradiation and then decreased with time. The uptake of[125 I]IMT was reduced to 87% and 60% of Day 0 at 1
and 2 days post-irradiation, respectively. The [125I]IMT
uptake decreased slightly with time. The uptake of [14C]
Met was reduced to 50% and 25% of Day 0 at 1 and 2days post-irradiation. Thereafter, the uptake of [14C]Met
remained unchanged.
3.2. In vivo experiments
Fig. 4 shows the time course of tumor volume changes
after irradiation using the carbon ion beam (25 Gy). The
tumor volumes continued to increase until 2 days after
irradiation and then began to shrink. On Day 4, the volumes
became significantly smaller (39%) compared to those at
Day 0. On Day 3, the uptake of [125 I]IMT was significantly
decreased compared to that on Day 0 (65%, Pb.05). A
significant reduction in [14C]Met uptake (41%, Pb.05) was
observed from Day 2 (Fig. 4C). No tumor regrowth wasobserved for one month (data not shown).
Fig. 5 depicts the uptakes of [125I]IMT and [14C]Met byirradiated or nonirradiated muscle for 5 days after irradiation
using the carbon ion beam (25 Gy). No significant

Fig. 6. SE-HPLC radiochromatograms of [14C]Met or [125I]IMT (top) and oftumor extracts 60 min after the injection of [14C]Met (A) and [125I]IMT (B)before (middle) or after (bottom) carbon ion beam irradiation. Under these
conditions, Met, IMT and free iodide had retention times of 25, 26, and 24
min, respectively.

differences were observed in [125 I]IMT and [14 C]Met
accumulation between irradiated and non-irradiated muscle.
Fig. 6 shows the SE-HPLC elution profiles of radioactivity from tumor extracts after injection of [125I]IMT or[14C]Met into mice before and 2 days after irradiation using
the carbon ion beam (25 Gy). The extracts of tumor
homogenates were obtained with radiochemical efficienciesof 85%. More than 90% of the radioactivity was detected as
intact [125I]IMT in irradiated and nonirradiated tumors. The
rest of the radioactivity was observed in fractions similar to
those of iodide. On the other hand, two additional
radioactivity peaks were observed at earlier retention times
after injection of [14C]Met in both irradiated and nonirradiated tumors. While the high molecular weight fractions
represented 51% of the total radioactivity in non-irradiatedFig. 5. Uptake of [125I]IMT (A) and [14C]Met (B) at irradiation sites (solid bar) after irradiation using the carbon ion beam (25 Gy) and at non-irradiated site
(hatched bar). Values are mean±S.D.

990

T. Uehara et al. / Nuclear Medicine and Biology 36 (2009) 985–991

tumors, the radioactivity of the fractions decreased to 43% of
the total radioactivity in irradiated tumors.

4. Discussion
The accumulation of [14C]Met in carbon ion beamirradiated tumor cells was reduced before morphologicalchanges (cell number and tumor volumes) of the tumor
became apparent (Figs. 3 and 4). These results were in good
agreement with the clinical studies reported previously[13,14]. The present studies also confirmed that the uptake ofboth [14C]Met and [125 I]IMT into tumor cells was mediatedby amino acid transport system L (Fig. 2). The accumulationof both [14C]Met and [125I]IMT at sites of irradiation was notobserved (Fig. 5). These findings supported the validity ofthis study design that the comparison of [14C]Met accumulation and metabolism with [125I]IMT in irradiated or
nonirradiated tumor cells could provide an insight forassessing the critical cellular reactions directly correlatedwith [11C]Met accumulation in tumors.
The reduction of both [14C]Met and [125 I]IMT occurred inadvance of the reduction of cell number and tumor volume(Figs. 3 and 4). This indicated that a dysfunction in aminoacid transport system L could be involved in the response oftumor cells to [14 C]Met uptake. However, while thereduction in [14C]Met accumulation in irradiated tumor
cells occurred 2 days after irradiation, [125I]IMT showed asignificant decrease in tumor accumulation 3 days after
irradiation (Figs. 3 and 4). This indicated that cellular
reactions other than amino acid transport system L could also
be involved in the tumor response to [14C]Met accumulation.
To further examine the cellular reactions, the metabolismof [14C]Met and [125 I]IMT in tumor cells was compared by
SE-HPLC before and 2 days after irradiation, because at 2days after irradiation of tumors using the carbon ion beam,
the tumor uptake of [14C]Met decreased, but that of [125I]
IMT was not changed (Fig. 4). The majority of [125I]IMT
remained intact in the tumor, and no changes in HPLC
radioactivity profiles were observed between irradiated andnon-irradiated tumors (Fig. 6). Contrary to [125 I]IMT, 14Cradioactivity was detected in both high-molecular-weightfractions and the intact [14C]Met fraction in tumors. In
addition, a decrease in the high molecular weight fraction
was observed in irradiated tumors (Fig. 6). A significant
reduction in the tumor accumulation of [14C]Met wasobserved at this postirradiation time (Fig. 4). It is wellknown that methionine is incorporated into protein synthesis
and that the methyl group of methionine is transferred toRNA, DNA, and polyamines in the cells [9,27]. These
intracellular processes are also known to proceed in anenergy-dependent manner [17]. On the other hand, it wasreported that, in mice given cycloheximide to inhibit protein
synthesis, the tumor uptake of [14C]leucine decreased morethan that of [3H]Met in tumor cells, although they were bothtransported by amino acid transporter system L [9]. Because

the uptake of Met reflects not only protein synthesis but also
other metabolic pathways, such transmethylation, the uptakeof Leu reflects protein synthesis. Thus, the findings in thisstudy implied that energy-dependent intracellular reactions
such as protein synthesis and trans-methylation could
constitute the key reactions that affect the changes in [11C]
Met accumulation in carbon ion beam-irradiated tumors.
Although the intracellular reactions are inherent to [11C]Met,
radiopharmaceuticals that reflect the activities of energydependent intracellular reactions may be useful for predicting the therapeutic efficacies.
In conclusion, the present study assessed the cellular
reactions directly correlated with the changes in [11C]Met
accumulation of the tumor cells. While the dysfunction ofamino acid transport system L was involved, energydependent intracellular activities could constitute the key
reactions that affect the radioactivity levels in the tumor cells
after injection of [11C]Met. These results also suggest thatradiopharmaceuticals that reflect the activities of energydependent intracellular reactions may be useful to predicttumor responses to carbon ion radiotherapy.
Acknowledgments
This study was supported in part by a Grant-in-Aid forYoung Scientists (B) and Special Funds for Education andResearch (Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and by the Special
Coordination Funds for Research Project with Heavy Ions at
the National Institute of Radiological Sciences-Heavy-ion
Medical Accelerator in Chiba (NIRS-HIMAC).
References[1] Kamada T, Tsujii H, Tsuji H, Yanagi T, Mizoe JE, Miyamoto T, et al.
Efficacy and safety of carbon ion radiotherapy in bone and soft tissue
sarcomas. J Clin Oncol 2002;20:4466–71.
[2] Tsujii H, Mizoe JE, Kamada T, Baba M, Kato S, Kato H, et al.
Overview of clinical experiences on carbon ion radiotherapy at NIRS.
Radiother Oncol 2004;73(Suppl 2):S41–9.
[3] Ando K, Koike S, Oohira C, Ogiu T, Yatagai F. Tumor induction inmice locally irradiated with carbon ions: a retrospective analysis. J
Radiat Res (Tokyo) 2005;46:185–90.
[4] Furusawa Y, Fukutsu K, Aoki M, Itsukaichi H, Eguchi-Kasai K, Ohara
H, et al. Inactivation of aerobic and hypoxic cells from three different
cell lines by accelerated 3He-, 12C- and 20Ne-ion beams. Radiat Res
2000;154:485–96.
[5] Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS,
Rubinstein L, et al. New guidelines to evaluate the response totreatment in solid tumors. European Organization for Research andTreatment of Cancer, National Cancer Institute of the United States,
National Cancer Institute of Canada. J Natl Cancer Inst 2000;92:
205–16.
[6] Kollmannsberger C, Oechsle K, Dohmen BM, Pfannenberg A, Bares
R, Claussen CD, et al. Prospective comparison of [18F]fluorodeoxyglucose positron emission tomography with conventional assessment
by computed tomography scans and serum tumor markers for theevaluation of residual masses in patients with nonseminomatous germ
cell carcinoma. Cancer 2002;94:2353–62.

T. Uehara et al. / Nuclear Medicine and Biology 36 (2009) 985–991[7] Nuutinen J, Sonninen P, Lehikoinen P, Sutinen E, Valavaara R, Eronen
E, et al. Radiotherapy treatment planning and long-term follow-up with
[11C]methionine PET in patients with low-grade astrocytoma. Int J
Radiat Oncol Biol Phys 2000;48:43–52.
[8] Saleem A, Charnley N, Price P. Clinical molecular imaging withpositron emission tomography. Eur J Cancer 2006;42:1720–7.
[9] Ishiwata K, Kubota K, Murakami M, Kubota R, Sasaki T, Ishii S, et al.
Re-evaluation of amino acid PET studies: can the protein synthesis
rates in brain and tumor tissues be measured in vivo? J Nucl Med 1993;
34:1936–43.
[10] Van de Wiele C, Lahorte C, Oyen W, Boerman O, Goethals I, Slegers
G, et al. Nuclear medicine imaging to predict response to radiotherapy:
a review. Int J Radiat Oncol Biol Phys 2003;55:5–15.
[11] Kubota K, Ishiwata K, Kubota R, Yamada S, Tada M, Sato T, et al.
Tracer feasibility for monitoring tumor radiotherapy: a quadruple tracer
study with fluorine-18-fluorodeoxyglucose or fluorine-18-fluorodeoxyuridine, L-[methyl-14C]methionine, [6-3H]thymidine, and gallium-67.
J Nucl Med 1991;32:2118–23.
[12] Kubota K, Matsuzawa T, Takahashi T, Fujiwara T, Kinomura S, Ido T,
et al. Rapid and sensitive response of carbon-11-L-methionine tumor
uptake to irradiation. J Nucl Med 1989;30:2012–6.
[13] Reinhardt MJ, Kubota K, Yamada S, Iwata R, Yaegashi H. Assessmentof cancer recurrence in residual tumors after fractionated radiotherapy:
a comparison of fluorodeoxyglucose, L-methionine and thymidine. J
Nucl Med 1997;38:280–7.
[14] Jager PL, Groen HJ, van der Leest A, van Putten JW, Pieterman RM,
de Vries EG, et al. L-3-[123I]iodo-alpha-methyl-tyrosine SPECT in
non-small cell lung cancer: preliminary observations. J Nucl Med
2001;42:579–85.
[15] Jager PL, Vaalburg W, Pruim J, de Vries EG, Langen KJ, Piers DA.
Radiolabeled amino acids: basic aspects and clinical applications inoncology. J Nucl Med 2001;42:432–45.
[16] Langen KJ, Muhlensiepen H, Holschbach M, Hautzel H, Jansen P,
Coenen HH. Transport mechanisms of 3-[123I]iodo-alpha-methyl-Ltyrosine in a human glioma cell line: comparison with [3H]methyl]-Lmethionine. J Nucl Med 2000;41:1250–5.
[17] Stern PH, Wallace CD, Hoffman RM. Altered methionine metabolism
occurs in all members of a set of diverse human tumor cell lines. J Cell
Physiol 1984;119:29–34.

991[18] Riemann B, Stogbauer F, Kopka K, Halfter H, Lasic M, Schirmacher
A, et al. Kinetics of 3-[123I]iodo-L-alpha-methyltyrosine transport in rat
C6 glioma cells. Eur J Nucl Med 1999;26:1274–8.
[19] Lahoutte T, Caveliers V, Dierickx L, Vekeman M, Everaert H,
Mertens J, et al. In vitro, characterization of the influx of 3-[125I]iodo125
L-alpha-methyltyrosine and 2-[ I]iodo-L-tyrosine into U266 human
myeloma cells: evidence for system T transport. Nucl Med Biol 2001;
28:129–34.
[20] Franzius C, Kopka K, van Valen F, Eckervogt V, Riemann B, Sciuk J,
et al. Characterization of 3-[123I]iodo-L-alpha-methyl tyrosine ([123I]
IMT) transport into human Ewing's sarcoma cells in vitro. Nucl Med
Biol 2001;28:123–8.
[21] Langen KJ, Pauleit D, Coenen HH. 3-[123I]Iodo-alpha-methyl-Ltyrosine: uptake mechanisms and clinical applications. Nucl Med Biol
2002;29:625–31.
[22] Tisljar U, Kloster G, Ritzl F, Stocklin G. Accumulation of radioiodinated L-alpha-methyltyrosine in pancreas of mice: concise
communication. J Nucl Med 1979;20:973–6.
[23] Langen KJ, Coenen HH, Roosen N, Kling P, Muzik O, Herzog H, et al.
SPECT studies of brain tumors with L-3-[123I] iodo-alpha-methyl
tyrosine: comparison with PET, 124IMT and first clinical results. J Nucl
Med 1990;31:281–6.
[24] Kawai K, Fujibayashi Y, Yonekura Y, Konishi J, Saji H, Kubodera A,
et al. An artificial amino acid radiopharmaceutical for single photon
emission computed tomographic study of pancreatic amino acidtransports 123I-3-iodo-alpha-methyl-L-tyrosine. Ann Nucl Med 1992;6:
169–75.
[25] Samnick S, Schaefer A, Siebert S, Richter S, Vollmar B, Kirsch CM.
Preparation and investigation of tumor affinity, uptake kinetic andtransport mechanism of iodine-123-labelled amino acid derivatives inhuman pancreatic carcinoma and glioblastoma cells. Nucl Med Biol
2001;28:13–23.
[26] Malo C. Free amino acid levels in serum and small intestine during thepost-natal development of normal and sparse-fur mutant mice. Comp
Biochem Physiol A Physiol 1994;109:1049–57.
[27] Ishiwata K, Ido T, Abe Y, Matsuzawa T, Iwata R. Tumor
uptake studies of S-adenosyl-L-[methyl-11C]methionine and L[methyl-11C]methionine. Int J Rad Appl Instrum B 1988;15:
123–6.